Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infections organisms) and internal factors (inherited mutations, hormones, immune conditions, and DNA damage). These factors may act together or sequentially to initiate and/or promote carcinogenesis. Cancer causes 1 of every 4 deaths and is the leading cause of death in people under age 85 in the United States. Nearly half of all men and a little over one third of all women in the U.S. will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer. The sooner a cancer is found and treatment begins, the better are the chances for living for many years.
Lung cancer is the leading cause of cancer deaths for both men and women in the United States. According to the American Cancer Society, about 160,000 people die annually of this disease, with about 170,000 newly diagnosed cases each year. Despite the use of surgery, chemotherapy, and radiation, the survival rate for patients remains extremely poor (>15% over 5 years). As estimated by the American Cancer Society, the 5-year survival rate for all cancers is about 64% for cancers diagnosed between 1995-2000. However, the survival rate varies depending on cancer type and the stage of cancer at time of detection. For example, the survival rate for brain, breast, and colon cancer is 33, 88, and 63%, respectively for cancers diagnosed between 1995-2000. Therefore, treatments in addition to the standard methods of treatment that include surgery, radiation, chemotherapy, immunotherapy, and hormone therapy are needed.
Misregulation of genes that control cell fate determination often contributes to cancer. Such altered genes are known as oncogenes. Oncogenes are called proto-oncogenes when they are normal (i.e., not mutated). Proto-oncogenes encode components of the cell's normal growth-control pathway. Some of these components are growth factors, receptors, signaling enzymes, and transcription factors.
Ras is one such oncogene. Mammalian ras genes code for closely related, small proteins (H-ras, K-ras and N-ras). Ras is found in normal cells, where it helps to relay signals by acting as a switch. When receptors on the cell surface are stimulated (by a hormone, for example), Ras is switched on and transduces signals that tell the cell to grow. If the cell-surface receptor is not stimulated, Ras is not activated and so the pathway that results in cell growth is not initiated. In about 30% of human cancers, Ras is mutated so that it is permanently switched on, telling the cell to grow regardless of whether receptors on the cell surface are activated or not. A high incidence of ras gene mutations is found in all lung cancers and adenocarcinomas (10% and 25%, respectively, K-ras), in malignant tumors of the pancreas (80-90%, K-ras), in colorectal carcinomas (30-60%, K-ras), in non-melanoma skin cancer (30-50%, H-ras), in hematopoietic neoplasia of myeloid origin (18-30%, K- and N-ras), and in seminoma (25-40%, K-ras). In other tumors, a mutant ras gene is found at a lower frequency: for example, in breast carcinoma (0-12%, K-ras), glioblastoma and neuroblastoma (0-10%, K- and N-ras).
Other oncogenes include members of the MYC family (c-MYC, N-MYC, and L-MYC), which have been widely studied, and amplification of myc genes has been found in a variety of tumor types including lung (c-MYC, N-MYC, L-MYC), colon (c-MYC), breast (c-MYC), and neuroblastoma (N-MYC). Genes that inhibit apoptosis have also been identified as oncogenes. The prototype of these genes is BCL-2. Originally identified at the chromosomal breakpoint in follicular lymphoma, this protein was found to inhibit cell death rather than promote cell growth. BCL-2 belongs to a family of intracellular proteins whose role is to regulate caspase activation that leads to DNA fragmentation and cell death. In melanoma, BCL-2 has been reported to be overexpressed in primary and metastatic lesions and this phenotype is associated with tumor progression.
Radiation therapy is one of the three primary modalities employed in cancer treatment. Although radiation has been in practice for over a century, the global genetic response necessary for tissues to survive radiation-induced injury remains largely unknown. This has limited the ability to develop meaningful routes to minimize normal tissue toxicity while enhancing tumor eradication. While single-protein targeting strategies have shown moderate success in preclinical models, few have been successful in human trials. Ras overexpression in tumors is considered a poor prognostic feature, and is hypothesized to be involved in the response to cytotoxic therapy. Ras signaling has been shown to be critical for protection from radiation induced target cell death (Brown and Wilson, Canc. Biol. & Therapy, 2:477-490 (2003)). Unfortunately, strategies directly targeting RAS or its upstream and/or downstream effectors have not successfully altered the radiation response in vivo.
A failure to identify radiation modulators may be due to the complex genetic cellular response to radiation, as indicated by microarray studies showing significant changes in the expression of at least 855 genes (>1.5 fold) within 4 hours of radiation. This suggests that regulatory molecules capable of regulating a large number of target genes in a rapid manner may be required to affect the radiation response.
Micro RNAs (referred to as “miRNAs”) are small non-coding RNAs, belonging to a class of regulatory molecules found in plants and animals that control gene expression by binding to complementary sites on target messenger RNA (mRNA) transcripts (SEQ ID Nos. 1, 2 and 3). miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs, which fold into imperfect stem-loop structures (Lee, Y., et al., Nature (2003) 425(6956):415-9) (FIG. 1). The pre-miRNAs undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer (Hutvagner, G., et al., Science (2001) 12:12 and Grishok, A., et al., Cell (2001) 106(1):23-34). mRNAs have been shown to regulate gene expression in two ways. First, miRNAs that bind to protein-coding mRNA sequences that are exactly complementary to the miRNA induce the RNA-mediated interference (RNAi) pathway. Messenger RNA targets are cleaved by ribonucleases in the RISC complex. This mechanism of miRNA-mediated gene silencing has been observed mainly in plants (Hamilton, A. J. and D. C. Baulcombe, Science (1999) 286(5441):950-2 and Reinhart, B. J., et al., MicroRNAs in plants. Genes and Dev. (2002) 16:1616-1626), but an example is known from animals (Yekta, S., I. H. Shih, and D. P. Bartel, Science (2004) 304(5670):594-6). In the second mechanism, miRNAs that bind to imperfect complementary sites on messenger RNA transcripts direct gene regulation at the posttranscriptional level but do not cleave their mRNA targets. mRNAs identified in both plants and animals use this mechanism to exert translational control of their gene targets (Bartel, D. P., Cell (2004) 116(2):281-97).
Hundreds of miRNAs have been identified in the fly, worm, plant and mammalian genomes. The biological role for the majority of the miRNAs remains unknown because almost all of these were found through cloning and bioinformatic approaches (Lagos-Quintana, M., et al., Curr Biol (2002) 12(9):735-9; Lagos-Quintana, M., et al., RNA (2003) 9(2): 175-179; Lagos-Quintana, M., et al., Science (2001) 294(5543): 853-8; Lee, R. C. and V. Ambros, Science (2001) 294(5543):862-4; Lau, N. C., et al., Science (2001) 294(5543):858-62; Lim, L. P., et al., Genes Dev (2003) 17(8):991-1008; Johnston, R. J. and O. Hobert, Nature (2003) 426(6968):845-9; and Chang, S., et al., Nature (2004) 430(7001):785-9).
It is likely that these uncharacterized miRNAs act as important gene regulators during development to coordinate proper organ formation, embryonic patterning, and body growth, but this remains to be established. In zebrafish, most miRNAs are expressed from organogenesis onward (Chen, P. Y., et al., Genes Dev (2005) 19(11):1288-93 and Wienholds, E., et al., Science, (2005)).
The biological roles for several miRNAs have been elucidated. These studies highlight the importance of these regulatory molecules in a variety of developmental and metabolic processes. For example, the Drosophila miRNA, bantam, was identified in a gain-of-function genetic screen for factors that caused abnormal tissue growth (Brennecke, J., et al., Cell (2003) 113(1):25-36). Bantam was found to induce tissue growth in the fly by both stimulating cell proliferation and inhibiting apoptosis (Brennecke, J., et al., Cell (2003). 113(1):25-36). Although the proliferation targets for bantam have not been identified, a pro-apoptotic gene, hid, was shown to have multiple bantam complementary sites in its 3′UTR. Since hid gene expression was repressed by the bantam miRNA, this implicates a role for bantam in controlling apoptosis by blocking hid function. Another Drosophila miRNA, mir-14, was identified in a genetic screen for factors that modified Reaper-induced apoptosis in the fly eye (Xu, P., et al., Curr Biol (2003) 13(9):790-5). mir-14 was shown to be a strong suppressor of apoptosis. In addition, mir-14 also appears to play a role in the Drosophila stress response as well as in regulating fat metabolism. mRNAs also regulate Notch pathway genes in Drosophila (Lai, et al. Genes Dev (2005) 19(9):1067-80). A mammalian miRNA, mir-181, was shown to direct the differentiation of human B cells (Chen, C. Z., et al., Science (2004) 303(5654): 83-6), mir-373 regulates insulin secretion (Poy, M. N., et al., Nature (2004) 432(7014):226-30), while other miRNAs regulate viral infections (Lecellier, C. H., et al., Science (2005) 308(5721):557-60 and Sullivan, C. S., et al., Nature (2005) 435(7042):682-6).
Studies to understand the mechanism of RNAi in C. elegans, Drosophila and human cells have shown that the miRNA and RNAi pathways may intersect (Grishok, A., et al. Cell (2001) 106(1):23-34 and Hutvagner, G., et al., Science (2001) 293(5531):834-8). mRNAs copurify with components of the RNAi effector complex, RISC, suggesting a link between miRNAs and siRNAs involved in RNAi (Mourelatos, Z., et al., Genes Dev (2002) 16(6):720-8; Hutvagner, G. and P. D. Zamore, Science (2002) 297(5589):2056-60; and Caudy, A. A., et al., Nature (2003) 425(6956): 411-4). There is also an indication that some protein factors may play a role in both the miRNA ribonucleoprotein (miRNP) and RISC and others might be unique to the miRNP (Grishok, A., et al., Cell (2001) 106(1): 23-34 and Carmell, M. A., et al., Genes Dev (2002) 16(21): 2733-42). For example, proteins of the argonaute/PAZ/PIWI family are components of both RISC and miRNPs. There is also mounting evidence that genes encoding these proteins are linked to cancer. hAgo3, hAgo1, and hAgo4 reside in region 1p34-35, often lost in Wilms' tumors, and Hiwi, is located on chromosome 12q24.33, which has been linked to the development of testicular germ cell tumors (Carmell, M. A., et al., Genes Dev (2002) 16(21):2733-42). In addition, DICER, the enzyme which processes miRNAs and siRNAs, is poorly expressed in lung cancers (Karube, Y., et al., Cancer Sci (2005) 96(2): 111-5).
It is therefore an object of the present invention to provide naturally occurring miRNAs for inhibition of expression of one or more oncogenes.
It is further an object of the present invention to provide naturally occurring nucleic acids for treatment or prophylaxis of one or more symptoms of cancer.
It is an even further object of the present invention to provide methods for sensitizing cancer cells to cytotoxic therapies including radiotherapy and chemotherapy.